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1.  Crystallization and preliminary X-ray diffraction of the surfactant protein Lv-ranaspumin from the frog Leptodactylus vastus  
The purification, crystallization and MS analysis of a natural surfactant protein from the frog L. vastus are described.
Lv-ranaspumin is a natural surfactant protein with a molecular mass of 23.5 kDa which was isolated from the foam nest of the frog Leptodactylus vastus. Only a partial amino-acid sequence is available for this protein and it shows it to be distinct from any protein sequence reported to date. The protein was purified from the natural source by ion-exchange and size-exclusion chromatography and was crystallized by sitting-drop vapour diffusion using the PEG/Ion screen at 293 K. A complete data set was collected to 3.5 Å resolution. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 51.96, b = 89.99, c = 106.00 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54%.
doi:10.1107/S1744309112002679
PMCID: PMC3310541  PMID: 22442233
Leptodactylus vastus; Lv-ranaspumin; surfactant proteins
2.  Crystallization and preliminary X-ray diffraction analysis of human dipeptidyl peptidase 10 (DPPY), a component of voltage-gated potassium channels 
The expression, purification and crystallization of human dipeptidyl peptidase 10, a component of voltage-gated potassium channels, is described.
Dipeptidyl peptidase 10 (DPP10, DPPY) is an inactive peptidase associated with voltage-gated potassium channels, acting as a modulator of their electrophysiological properties, cell-surface expression and subcellular localization. Because potassium channels are important disease targets, biochemical and structural characterization of their interaction partners was sought. DPP10 was cloned and expressed using an insect-cell system and the protein was purified via His-tag affinity and size-exclusion chromatography. Crystals obtained by the sitting-drop method were orthorhombic, belonging to space group P212121 with unit-cell parameters a = 80.91, b = 143.73, c = 176.25 Å. A single solution with two molecules in the asymmetric unit was found using the structure of DPP6 (also called DPPX; PDB entry 1xfd) as the search model in a molecular replacement protocol.
doi:10.1107/S1744309111055230
PMCID: PMC3274407  PMID: 22298003
dipeptidyl peptidase 10; voltage-gated potassium channels
3.  Structures of Human DPP7 Reveal the Molecular Basis of Specific Inhibition and the Architectural Diversity of Proline-Specific Peptidases 
PLoS ONE  2012;7(8):e43019.
Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/β-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The “specificity domains” are structurally also completely different exhibiting a β-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.
doi:10.1371/journal.pone.0043019
PMCID: PMC3430648  PMID: 22952628
4.  Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia boliviana Piper seeds 
Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained using 0.1 M HEPES pH 7.5 and 3.0 M sodium formate.
Plant lectins are the most studied group of carbohydrate-binding proteins. Despite the high similarity between the members of the Diocleinae subtribe (Leguminosae) group, they present differing biological activities. Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained under the condition 0.1 M HEPES pH 7.5 and 3.0 M sodium formate. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 126.70, b = 66.64, c = 64.99 Å, α = 90.0, β = 120.8, γ = 90.0°. Assuming the presence of a dimer in the asymmetric unit, the solvent content was estimated to be about 46%. A complete data set was collected at 1.5 Å resolution.
doi:10.1107/S1744309109000797
PMCID: PMC2650465  PMID: 19255467
lectins; Canavalia boliviana Piper
5.  New crystal forms of Diocleinae lectins in the presence of different dimannosides 
The crystallization and preliminary X-­ray data of Canavalia gladiata lectin (CGL) and C. maritima lectin (CML) complexed with Man(α1-2)Man(α1)OMe, Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe in two crystal forms [the complexes with Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe crystallized in space group P32 and those with Man(α1-2)Man(α1)OMe crystallized in space group I222], which differed from those of the native proteins (P21212 for CML and C222 for CGL), are reported.
Studying the interactions between lectins and sugars is important in order to explain the differences observed in the biological activities presented by the highly similar proteins of the Diocleinae subtribe. Here, the crystallization and preliminary X-­ray data of Canavalia gladiata lectin (CGL) and C. maritima lectin (CML) complexed with Man(α1-2)Man(α1)OMe, Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe in two crystal forms [the complexes with Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe crystallized in space group P32 and those with Man(α1-2)Man(α1)OMe crystallized in space group I222], which differed from those of the native proteins (P21212 for CML and C222 for CGL), are reported. The crystal complexes of ConA-like lectins with Man(α1-4)Man(α1)OMe are reported here for the first time.
doi:10.1107/S1744309106038887
PMCID: PMC2225211  PMID: 17077488
lectin–sugar interactions; Dioocleinae lectins
6.  Crystallization and preliminary X-ray diffraction analysis of the lectin from Dioclea rostrata Benth seeds 
D. rostrata lectin was crystallized by hanging-drop vapor diffusion. The crystal belongs to the orthorhombic space group I222 and diffracted to 1.87 Å resolution.
Lectins from the Diocleinae subtribe (Leguminosae) are highly similar proteins that promote various biological activities with distinctly differing potencies. The structural basis for this experimental data is not yet fully understood. Dioclea rostrata lectin was purified and crystallized by hanging-drop vapour diffusion at 293 K. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 61.51, b = 88.22, c = 87.76 Å.  Assuming the presence of one monomer per asymmetric unit, the solvent content was estimated to be about 47.9%. A complete data set was collected at 1.87 Å resolution.
doi:10.1107/S1744309106001801
PMCID: PMC2150952  PMID: 16511292
lectins; Dioclea rostrata

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